The present invention relates to nucleosidic monomers and oligomeric compounds incorporating such nucleosidic monomers, and methods of using such oligomeric compounds. The oligomeric compounds of the invention are useful for therapeutic and investigative purposes. More specifically, the present invention is directed to the use of oligomeric compounds having 2xe2x80x2-O-modifications that will increase their affinity and nuclease resistance.
It is well known that most of the bodily states in mammals, including most disease states, are affected by proteins. Classical therapeutic modes have generally focused on interactions with such proteins in an effort to moderate their disease-causing or disease-potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, such as intracellular RNA. By interfering with the production of proteins, maximum therapeutic effect and minimal side effects may be realized. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides. Oligonucleotides are now accepted as therapeutic agents with great promise, and are known to hybridize to single-stranded DNA or RNA molecules. Hybridization is the sequence-specific base pair hydrogen bonding of nucleobases of the oligonucleotide to the nucleobases of the target DNA or RNA molecule. Such nucleobase pairs are said to be complementary to one another. The concept of inhibiting gene expression through the use of sequence-specific binding of oligonucleotides to target RNA sequences, also known as antisense inhibition, has been demonstrated in a variety of systems, including living cells (see, e.g., Wagner et al., Science (1993) 260:1510-1513; Milligan et al., J. Med. Chem., (1993) 36:1923-37; Uhlmann et al., Chem. Reviews, (1990) 90:543-584; Stein et al., Cancer Res., (1988) 48:2659-2668).
The events that provide the disruption of the nucleic acid function by antisense oligonucleotides (Cohen in Oligonucleotides: Antisense Inhibitors of Gene Expression, (1989) CRC Press, Inc., Boca Raton, Fla.) are thought to be of two types. The first, hybridization arrest, denotes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides: Miller, P. S. and Ts""O, P.O.P. (1987) Anti-Cancer Drug Design, 2:117-128, and xcex1-anomer oligonucleotides are the two most extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
The second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of the targeted RNA by intracellular RNase H. A 2xe2x80x2-deoxyribofuranosyl oligonucleotide or oligonucleotide analog hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are the most prominent example of an antisense agent that operates by this type of antisense terminating event.
Oligonucleotides may also bind to duplex nucleic acids to form triplex complexes in a sequence specific manner via Hoogsteen base pairing (Beal et al., Science, (1991) 251:1360-1363; Young et al., Proc. Natl. Acad. Sci. (1991) 88:10023-10026). Both antisense and triple helix therapeutic strategies are directed towards nucleic acid sequences that are involved in or responsible for establishing or maintaining disease conditions. Such target nucleic acid sequences may be found in the genomes of pathogenic organisms including bacteria, yeasts, fungi, protozoa, parasites, viruses, or may be endogenous in nature. By hybridizing to and modifying the expression of a gene important for the establishment, maintenance or elimination of a disease condition, the corresponding condition may be cured, prevented or ameliorated.
In determining the extent of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of an oligonucleotide to bind to the complementary nucleic acid may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm), a characteristic physical property of double helices, denotes the temperature (in degrees centigrade) at which 50% helical (hybridized) versus coil (unhybridized) forms are present. Tm is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the bonds between the strands.
Oligonucleotides may also be of therapeutic value when they bind to non-nucleic acid biomolecules such as intracellular or extracellular polypeptides, proteins, or enzymes. Such oligonucleotides are often referred to as xe2x80x9captamersxe2x80x9d and they typically bind to and interfere with the function of protein targets (Griffin, et al., Blood, (1993), 81:3271-3276; Bock, et al., Nature, (1992) 355: 564-566).
Oligonucleotides and their analogs (oligomeric compounds) have been developed and used for diagnostic purposes, therapeutic applications and as research reagents. For use as therapeutics, oligonucleotides preferably are transported across cell membranes or be taken up by cells, and appropriately hybridize to target DNA or RNA. These functions are believed to depend on the initial stability of the oligonucleotides toward nuclease degradation. A deficiency of unmodified oligonucleotides which affects their hybridization potential with target DNA or RNA for therapeutic purposes is their degradation by a variety of ubiquitous intracellular and extracellular nucleolytic enzymes referred to as nucleases. For oligonucleotides to be useful as therapeutics or diagnostics, the oligonucleotides should demonstrate enhanced binding affinity to complementary target nucleic acids, and preferably be reasonably stable to nucleases and resist degradation. For a non-cellular use such as a research reagent, oligonucleotides need not necessarily possess nuclease stability.
A number of chemical modifications have been introduced into oligonucleotides to increase their binding affinity to target DNA or RNA and resist nuclease degradation. Modifications have been made, for example, to the phosphate backbone to increase the resistance to nucleases. These modifications include use of linkages such as methyl phosphonates, phosphorothioates and phosphorodithioates, and the use of modified sugar moieties such as 2xe2x80x2-O-alkyl ribose. Other oligonucleotide modifications include those made to modulate uptake and cellular distribution. A number of modifications that dramatically alter the nature of the internucleotide linkage have also been reported in the literature. These include non-phosphorus linkages, peptide nucleic acids (PNA""s) and 2xe2x80x2-5xe2x80x2 linkages. Another modification to oligonucleotides, usually for diagnostic and research applications, is labeling with non-isotopic labels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules.
Over the last ten years, a variety of synthetic modifications have been proposed to increase nuclease resistance, or to enhance the affinity of the antisense strand for its target mRNA (Crooke et al., Med. Res. Rev., 1996, 16, 319-344; De Mesmaeker et al., Acc. Chem. Res., 1995, 28, 366-374). A variety of modified phosphorus-containing linkages have been studied as replacements for the natural, readily cleaved phosphodiester linkage in oligonucleotides. In general, most of them, such as the phosphorothioate, phosphoramidates, phosphonates and phosphorodithioates all result in oligonucleotides with reduced binding to complementary targets and decreased hybrid stability.
RNA exists in what has been termed xe2x80x9cA Formxe2x80x9d geometry while DNA exists in xe2x80x9cB Formxe2x80x9d geometry. In general, RNA:RNA duplexes are more stable, or have higher melting temperatures (Tm) than DNA:DNA duplexes (Sanger et al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634). The increased stability of RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2xe2x80x2 hydroxyl in RNA biases the sugar toward a C3xe2x80x2 endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry. On the other hand, deoxy nucleic acids prefer a C2xe2x80x2 endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, N.Y.). In addition, the 2xe2x80x2 hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al., Biochemistry, 1996, 35, 8489-8494).
DNA:RNA hybrid duplexes, however, are usually less stable than pure RNA:RNA duplexes and, depending on their sequence, may be either more or less stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The structure of a hybrid duplex is intermediate between A- and B-form geometries, which may result in poor stacking interactions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306; Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996, 264, 521-533). The stability of a DNA:RNA hybrid a significant aspect of antisense therapies, as the proposed mechanism requires the binding of a modified DNA strand to a mRNA strand. Ideally, the antisense DNA should have a very high binding affinity with the mRNA. Otherwise, the desired interaction between the DNA and target mRNA strand will occur infrequently, thereby decreasing the efficacy of the antisense. oligonucleotide.
One synthetic 2xe2x80x2-modification that imparts increased nuclease resistance and a very high binding affinity to nucleotides is the 2xe2x80x2-methoxyethoxy (MOE, 2xe2x80x2-OCH2CH2OCH3) side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000; Freier et al., Nucleic Acids Res., 1997, 25, 4429-4443). One of the immediate advantages of the MOE substitution is the improvement in binding affinity, which is greater than many similar 2xe2x80x2 modifications such as O-methyl, O-propyl, and O-aminopropyl (Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443). 2xe2x80x2-O-Methoxyethyl-substituted also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926). Relative to DNA, they display improved RNA affinity and higher nuclease resistance. Chimeric oligonucleotides with 2xe2x80x2-O-methoxyethyl-ribonucleoside wings and a central DNA-phosphorothioate window also have been shown to effectively reduce the growth of tumors in animal models at low doses. MOE substituted oligonucleotides have shown outstanding promise as antisense agents in several disease states. One such MOE substituted oligonucleotide is presently being investigated in clinical trials for the treatment of CMV retinitis.
Although the known modifications to oligonucleotides, including the use of the 2xe2x80x2-O-methoxyethyl modification, have contributed to the development of oligonucleotides for various uses, there still exists a need in the art for further modifications that will impart enhanced hybrid binding affinity and/or increased nuclease resistance to oligonucleotides and their analogs.
The present invention provides oligomeric compounds having at least one 2xe2x80x2xe2x80x94Oxe2x80x94CH2CH2xe2x80x94Oxe2x80x94CH2CH2xe2x80x94N (R1)(R2) modified nucleoside. Preferred oligomeric compounds of the invention are those that include at least one nucleoside of the formula: 
wherein
Bx is a heterocyclic base;
each R1 and R2 is, independently, H, a nitrogen protecting group, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, wherein said substitution is OR3, SR3, NH3+, N(R3)(R4), guanidino or acyl where said acyl is an acid, amide or an ester;
or R1 and R2, together, are a nitrogen protecting group or are joined in a ring structure that optionally includes an additional heteroatom selected from N and O; and
each R3 and R4 is, independently, H, C1-C10 alkyl, a nitrogen protecting group, or R3 and R4, together, are a nitrogen protecting group;
or R3 and R4 are joined in a ring structure that optionally includes an additional heteroatom selected from N and O.
In one embodiment R1 is H, C1-C10 alkyl or C1-C10 substituted alkyl and R2 is C1-C10 substituted alkyl. In another embodiment R1 is C1-C10 alkyl. In a further embodiment R2 is C1-C10 substituted alkyl and the substituent is NH3+ or N(R3)(R4). In another embodiment R1 and R2 are both C1-C10 substituted alkyl with preferred substituents independently selected from NH3+ and N(R3)(R4). In yet a further embodiment both R1 and R2 are C1-C10 alkyl.
In one embodiment R1 and R2 are joined in a ring structure that can include at least one heteroatom selected from N and O. Preferred ring structures are imidazole, piperidine, morpholine or a substituted piperazine with a preferred substituent being C1-C12 alkyl.
In one embodiment the heterocyclic base is a purine or a pyrimidine with preferred heterocyclic bases being adenine, cytosine, 5-methylcytosine, thymine, uracil, guanine or 2-aminoadenine.
In one embodiment the oligomeric compound comprises from about 5 to about 50 nucleosides. In a preferred embodiment the oligomeric compound comprises from about 8 to about 30 nucleosides with a preferred range from about 15 to about 25 nucleosides.
The present invention also includes nucleosidic compounds of the formula: 
wherein:
Bx is a heterocyclic base;
T1 and T2, independently, are OH, a protected hydroxyl, an activated phosphorus group, a reactive group for forming an internucleotide linkage, a nucleoside, a nucleotide, an oligonucleoside an oligonucleotide or a linkage to a solid support;
each R1 and R2 is, independently, H, a nitrogen protecting group, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, wherein said substitution is OR3, SR3, NH3+, N (R3)(R4), guanidino or acyl where said acyl is an acid, amide or an ester;
or R1 and R2, together, are a nitrogen protecting group or are joined in a ring structure that optionally includes an additional heteroatom selected from N and O; and
each R3 and R4 is, independently, H, C1-C10 alkyl, a nitrogen protecting group, or R3 and R4, together, are a nitrogen protecting group;
or R3 and R4 are joined in a ring structure that optionally includes an additional heteroatom selected from N and O.
In one embodiment R1 is H, C1-C10 alkyl or C1-C10 substituted alkyl and R2 is C1-C10 substituted alkyl. In another embodiment R1 is C1-C10 alkyl. In a further embodiment R2 is C1-C10 substituted alkyl and the substituent is NH3+ or N (R3)(R4). In another embodiment R1 and R2 are both C1-C10 substituted alkyl with preferred substituents independently selected from NH3+ and N (R3)(R4). In yet a further embodiment both R1 and R2 are C1-C10 alkyl.
In one embodiment R1 and R2 are joined in a ring structure that can include at least one heteroatom selected from N and O. Preferred ring structures are imidazole, piperidine, morpholine or a substituted piperazine with a preferred substituent being C1-C12 alkyl.
In one embodiment the heterocyclic base is a purine or a pyrimidine with preferred heterocyclic bases being adenine, cytosine, 5-methylcytosine, thymine, uracil, guanine or 2-aminoadenine.
In one embodiment T1is a hydroxyl protecting group. In another embodiment T2 is an activated phosphorus group or a connection to a solid support. A preferred solid support material is microparticles. With CPG being a more preferred solid support material.
The present invention is directed to novel 2xe2x80x2-O-modified nucleosidic monomers and to oligomeric compounds incorporating these novel 2xe2x80x2-O-modified nucleosidic monomers. These modifications have certain desirable properties that contribute toward increases in binding affinity and/or nuclease resistance.
There are a number of items to consider when designing oligomeric compounds having enhanced binding affinities. One effective approach to constructing oligomeric compounds with very high RNA binding affinity relates to the combination of two or more different types of modifications, each of which contributes favorably to various factors that might be important for binding affinity.
Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443, recently published a study on the influence of structural modifications of oligonucleotides on the stability of their duplexes with target RNA. In this study, the authors reviewed a series of oligonucleotides containing more than 200 different modifications that had been synthesized and assessed for their hybridization affinity and Tm. Sugar modifications studied included substitutions on the 2xe2x80x2-position of the sugar, 3xe2x80x2-substitution, replacement of the 4xe2x80x2-oxygen, the use of bicyclic sugars, and four member ring replacements. Several heterocyclic base modifications were also studied including substitutions at the 5, or 6 position of thymine, modifications of pyrimidine heterocycles and modifications of purine heterocycles. Numerous backbone modifications were also investigated including backbones bearing phosphorus, backbones that did not bear a phosphorus atom, and backbones that were neutral.
Four general approaches potentially may be used to improve hybridization of oligonucleotides to RNA targets. These include: preorganization of the sugars and phosphates of the oligodeoxynucleotide strand into conformations favorable for hybrid formation, improving stacking of nucleobases by the addition of polarizable groups to the heterocycle bases of the nucleosidic monomers of the oligonucleotide, increasing the number of H-bonds available for A-U pairing, and neutralization of backbone charge to facilitate removing undesirable repulsive interactions. This invention principally employs the first of these, preorganization of the sugars and phosphates of the oligodeoxynucleotide strand into conformations favorable for hybrid formation, and can be used in combination with the other three approaches.
Sugars in DNA:RNA hybrid duplexes frequently adopt a C3xe2x80x2 endo conformation. Thus, modifications that shift the conformational equilibrium of the sugar moieties in the single strand toward this conformation should preorganize the antisense strand for binding to RNA. Of the several sugar modifications that have been reported and studied in the literature, the incorporation of electronegative substituents such as 2xe2x80x2-fluoro or 2xe2x80x2-alkoxy shift the sugar conformation towards the 3xe2x80x2 endo (northern) pucker conformation. This pucker conformation further assisted in increasing the Tm of the oligonucleotide with its target.
There is a clear correlation between substituent size at the 2xe2x80x2-position and duplex stability. Incorporation of alkyl substituents at the 2xe2x80x2-position typically leads to a significant decrease in binding affinity. Thus, small alkoxy groups generally are very favorable while larger alkoxy groups at the 2xe2x80x2-position generally are unfavorable. However, if the 2xe2x80x2-substituent contained an ethylene glycol motif, then a strong improvement in binding affinity to the target RNA is observed.
The high binding affinity resulting from 2xe2x80x2-substitution has been partially attributed to the 2xe2x80x2-substitution causing a C3xe2x80x2 endo sugar pucker which in turn may give the oligomer a favorable A-form like geometry. This is a reasonable hypothesis since substitution at the 2xe2x80x2 position by a variety of electronegative groups (such as fluoro and O-alkyl chains) has been demonstrated to cause C3xe2x80x2 endo sugar puckering (De Mesmaeker et al., Acc. Chem. Res., 1995, 28, 366-374; Lesnik et al., Biochemistry, 1993, 32, 7832-7838).
In addition, for 2xe2x80x2-substituents containing an ethylene glycol motif, a gauche interaction between the oxygen atoms around the Oxe2x80x94Cxe2x80x94Cxe2x80x94O torsion of the side chain may have a stabilizing effect on the duplex (Freier et al., Nucleic Acids Research, (1997) 25:4429-4442). Such gauche interactions have been observed experimentally for a number of years (Wolfe et al., Acc. Chem. Res., 1972, 5, 102; Abe et al., J. Am. Chem. Soc., 1976, 98, 468). This gauche effect may result in a configuration of the side chain that is favorable for duplex formation. The exact nature of this stabilizing configuration has not yet been explained. While we do not want to be bound by theory, it may be that holding the Oxe2x80x94Cxe2x80x94Cxe2x80x94O torsion in a single gauche configuration, rather than a more random distribution seen in an alkyl side chain, provides an entropic advantage for duplex formation.
The present invention has 2xe2x80x2 side chain having the formula: 2xe2x80x2xe2x80x94OCH2CH2OCH2CH2N (R1)(R2), where R1 and R2 can each be alkyl or substituted alkyl groups which gives a tertiary amine capable of being protonated. When R1 and R2 are both methyl groups the pKa of the side chain is 9.0 to 10.0 (aliphatic saturated 3xc2x0 amine). This tertiary amine is expected to be protonated at physiological pH (7.0), and in endosomes and lysosomes (pH 5.0). The resulting positive charge should improve the biostability of the drug by either inhibiting the nuclease from binding to the oligonucleotide or displacing the metal ions needed for the nucleases to carry on their function (Beese et al., EMBO J., 1991, 10, 25-33; and Brautigam et al., J. Mol. Bio., 1998, 277, 363-377).
As used herein, the term oligonucleoside includes oligomers or polymers containing two or more nucleoside subunits having a non-phosphorous linking moiety. Oligonucleosides according to the invention are monomeric subunits having a ribofuranose moiety attached to a heterocyclic base via a glycosyl bond. An oligonucleotide/nucleoside for the purposes of the present invention is a mixed backbone oligomer having at least two nucleosides covalently bound by a non-phosphate linkage and at least one phosphorous containing covalent bond with a nucleotide, and wherein at least one of the monomeric nucleotide or nucleoside units is a 2xe2x80x2-O-substituted compound prepared using the process of the present invention. An oligo-nucleotide/nucleoside can additionally have a plurality of nucleotides and nucleosides coupled through phosphorous containing and/or non-phosphorous containing linkages.
In the context of this invention, the term xe2x80x9coligomeric compoundxe2x80x9d refers to a plurality of nucleosides joined together in a specific sequence from naturally and non-naturally occurring nucleosides. The term includes oligonucleotides, oligonucleotide analogs, oligonucleosides having non-phosphorus containing internucleoside linkages and chimeric oligomeric compounds having mixed internucleoside linkages which can include all phosphorus or phosphorus and non-phosphorus containing internucleoside linkages. Each of the oligomeric compounds of the invention have at least one modified nucleoside where the modification is an aminooxy compound of the invention. Preferred nucleosides of the invention are joined through a sugar moiety via phosphorus linkages, and include adenine, guanine, adenine, cytosine, uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, other aza and deaza thymidines, other aza and deaza cytosines, other aza and deaza adenines, other aza and deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.
phosphorus containing linkages
phosphorodithioate (xe2x80x94Oxe2x80x94P(S)(S)xe2x80x94Oxe2x80x94);
phosphorothioate (xe2x80x94Oxe2x80x94P(S)(O)xe2x80x94Oxe2x80x94);
phosphoramidate (xe2x80x94Oxe2x80x94P(O)(NJ)xe2x80x94Oxe2x80x94);
phosphonate (xe2x80x94Oxe2x80x94P(J)(O)xe2x80x94Oxe2x80x94);
phosphotriesters (xe2x80x94Oxe2x80x94P(O J)(O)xe2x80x94Oxe2x80x94);
phophosphoramidate (xe2x80x94Oxe2x80x94P(O)(NJ)xe2x80x94Sxe2x80x94);
thionoalkylphosphonate (xe2x80x94Oxe2x80x94P(S)(J)xe2x80x94Oxe2x80x94);
thionoalkylphosphotriester (xe2x80x94Oxe2x80x94P(O)(OJ)xe2x80x94Sxe2x80x94);
boranophosphate (xe2x80x94R5xe2x80x94P(O)(O)xe2x80x94Jxe2x80x94);
non-phosphorus containing linkages
thiodiester (xe2x80x94Oxe2x80x94C(O)xe2x80x94Sxe2x80x94);
thionocarbamate (xe2x80x94Oxe2x80x94C(O)(NJ)xe2x80x94Sxe2x80x94);
siloxane (xe2x80x94Oxe2x80x94Si(J)2xe2x80x94Oxe2x80x94);
carbamate (xe2x80x94Oxe2x80x94C(O)xe2x80x94NHxe2x80x94 and xe2x80x94NHxe2x80x94C(O)xe2x80x94Oxe2x80x94)
sulfamate (xe2x80x94Cxe2x80x94S(O)(O)xe2x80x94Nxe2x80x94 and xe2x80x94Nxe2x80x94S(O)(O)xe2x80x94Nxe2x80x94;
morpholino sulfamide (xe2x80x94Oxe2x80x94S(O)(N(morpholino)xe2x80x94);
sulfonamide (xe2x80x94Oxe2x80x94SO2xe2x80x94NHxe2x80x94);
sulfide (xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94);
sulfonate (xe2x80x94Oxe2x80x94SO2xe2x80x94CH2xe2x80x94);
N,Nxe2x80x2-dimethylhydrazine (xe2x80x94CH2xe2x80x94N(CH3)xe2x80x94N(CH3)xe2x80x94);
thioformacetal (xe2x80x94Sxe2x80x94CH2xe2x80x94Oxe2x80x94);
formacetal (xe2x80x94Oxe2x80x94CH2xe2x80x94Oxe2x80x94);
thioketal (xe2x80x94Sxe2x80x94C(J)2xe2x80x94Oxe2x80x94); and
ketal (xe2x80x94Oxe2x80x94C(J)2xe2x80x94Oxe2x80x94);
amine (xe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94);
hydroxylamine (xe2x80x94CH2xe2x80x94N(J)xe2x80x94Oxe2x80x94);
hydroxylimine (xe2x80x94CHxe2x95x90Nxe2x80x94Oxe2x80x94); and
hydrazinyl (xe2x80x94CH2xe2x80x94N(H)xe2x80x94N(H)xe2x80x94).
wherexe2x80x9cJxe2x80x9d denotes a substituent group which is commonly hydrogen or an alkyl group or a more complicated group that varies from one type of linkage to another.
In addition to linking groups as described above that involve the modification or substitution of the xe2x80x94Oxe2x80x94Pxe2x80x94Oxe2x80x94 atoms of a naturally occurring linkage, included within the scope of the present invention are linking groups that include modification of the 5xe2x80x2-methylene group as well as one or more of the xe2x80x94Oxe2x80x94Pxe2x80x94Oxe2x80x94 atoms. Linkages of this type are well documented in the prior art and include without limitation the following:
amides (xe2x80x94CH2xe2x80x94CH2xe2x80x94N(H)xe2x80x94C(O)) and xe2x80x94CH2xe2x80x94Oxe2x80x94Nxe2x95x90CHxe2x80x94; and
alkylphosphorus (xe2x80x94C(J)2xe2x80x94P(xe2x95x90O)(OJ)xe2x80x94C(J)2xe2x80x94C(J)2xe2x80x94).
wherein J is as described above.
Synthetic schemes for the synthesis of the substitute internucleoside linkages described above are disclosed in: WO 91/08213; WO 90/15065; WO 91/15500; WO 92/20822; WO 92/20823; WO 91/15500; WO 89/12060; EP 216860; U.S. Pat. Nos. 9,204,294; 9,003,138; 9,106,855; 9,203,385; 9,103,680; U.S. Pat. Ser. Nos. 07/990,848; 07/892,902; 07/806,710; 07/763,130; 07/690,786; U.S. Pat. Nos. 5,466,677; 5,034,506; 5,124,047; 5,278,302; 5,321,131; 5,519,126; 4,469,863; 5,455,233; 5,214,134; 5,470,967; 5,434,257; Stirchak, E. P., et al., Nucleic Acid Res., 1989, 17, 6129-6141; Hewitt, J. M., et al., 1992, 11, 1661-1666; Sood, A., et al., J. Am. Chem. Soc., 1990, 112, 9000-9001; Vaseur, J. J. et al., J. Amer. Chem. Soc., 1992, 114, 4006-4007; Musichi, B., et al., J. Org. Chem., 1990, 55, 4231-4233; Reynolds, R. C., et al., J. Org. Chem., 1992, 57, 2983-2985; Mertes, M. P., et al., J. Med. Chem., 1969, 12, 154-157; Mungall, W. S., et al., J. Org. Chem., 1977, 42, 703-706; Stirchak, E. P., et al., J. Org. Chem., 1987, 52, 4202-4206; Coull, J. M., et al., Tet. Lett., 1987, 28, 745; and Wang, H., et al., Tet. Lett., 1991, 32, 7385-7388.
The nucleosidic monomers and oligomeric compounds of the invention can include modified sugars and modified bases (see, e.g., U.S. Pat. No. 3,687,808 and PCT application PCT/US89/02323). Such oligomeric compounds are best described as being structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic wild type oligonucleotides. Representative modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at their 2xe2x80x2 position, sugars having substituent groups at their 3xe2x80x2 position, and sugars having substituents in place of one or more hydrogen atoms of the sugar. Representative modifications are disclosed in International Publication Numbers WO 91/10671, published Jul. 25, 1991, WO 92/02258, published Feb. 20, 1992, WO 92/03568, published Mar. 5, 1992, and U.S. Pat. Nos. 5,138,045, 5,218,105, 5,223,618 5,359,044, 5,378,825, 5,386,023, 5,457,191, 5,459,255, 5,489,677, 5,506,351, 5,541,307, 5,543,507, 5,571,902, 5,578,718, 5,587,361, 5,587,469, all assigned to the assignee of this application. The disclosures of each of the above referenced publications are herein incorporated by reference.
Additional modifications may also be made at for example the 3xe2x80x2 position of the sugar on the 3xe2x80x2 terminal nucleosidic monomer and the 5xe2x80x2 position of the 5xe2x80x2 terminal nucleosidic monomer. In one aspect of the invention moieties or conjugates which enhance activity, cellular distribution or cellular uptake are chemically linked to one or more positions that are available for modification. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides and nucleosidic monomers, 1995, 14, 969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).
Representative heterocyclic bases amenable to the present invention include guanine, cytosine, uridine, and thymine, as well as other synthetic and natural nucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley and Sons, 1990, and those disclosed by Englisch, et al., Angewandte Chemie, International Edition 1991, 30, 613. All such oligomeric compounds are comprehended by this invention.
The nucleosidic monomers used in preparing oligomeric compounds of the present invention can include appropriate activated phosphorus groups such as activated phosphate groups and activated phosphite groups. As used herein, the terms activated phosphate and activated phosphite groups refer to activated monomers or oligomers that are reactive with a hydroxyl group of another monomeric or oligomeric compound to form a phosphorus-containing internucleotide linkage. Such activated phosphorus groups contain activated phosphorus atoms in pIII or pV valency states. Such activated phosphorus atoms are known in the art and include, but are not limited to, phosphoramdite, H-phosphonate and phosphate triesters. A preferred synthetic solid phase synthesis utilizes phosphoramidites as activated phosphates. The phosphoramidites utilize PIII chemistry. The intermediate phosphite compounds are subsequently oxidized to the PV state using known methods to yield, in a preferred embodiment, phosphodiester or phosphorothioate internucleotide linkages. Additional activated phosphates and phosphites are disclosed in Tetrahedron Report Number 309 (Beaucage and Iyer, Tetrahedron, 1992, 48, 2223-2311).
A number of chemical functional groups can be introduced into compounds of the invention in a blocked form and subsequently deblocked to form a final, desired compound. Such groups can be introduced as groups directly or indirectly attached at the heterocyclic base and the sugar substituents at the 2xe2x80x2, 3xe2x80x2 and 5xe2x80x2-positions. In general, a blocking group renders a chemical functionality of a larger molecule inert to specific reaction conditions and can later be removed from such functionality without substantially damaging the remainder of the molecule (Green and Wuts, Protective Groups in Organic Synthesis, 2d edition, John Wiley and Sons, New York, 1991). For example, the nitrogen atom of amino groups can be blocked as phthalimido groups, as 9-fluorenylmethoxycarbonyl (FMOC) groups, and with triphenylmethylsulfenyl, t-BOC or benzyl groups. Carboxyl groups can be protected as acetyl groups. Representative hydroxyl protecting groups are described by Beaucage et al., Tetrahedron 1992, 48, 2223. Preferred hydroxyl protecting groups are acid-labile, such as the trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). Chemical functional groups can also be xe2x80x9cblockedxe2x80x9d by including them in a precursor form. Thus, an azido group can be used considered as a xe2x80x9cblockedxe2x80x9d form of an amine since the azido group is easily converted to the amine. Further representative protecting groups utilized in oligonucleotide synthesis are discussed in Agrawal, et al., Protocols for Oligonucleotide Conjugates, Eds, Humana Press; New Jersey, 1994; Vol. 26 pp. 1-72.
Examples of hydroxyl protecting groups include, but are not limited to, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p,pxe2x80x2-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate, chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate, p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate.
Amino-protecting groups stable to acid treatment are selectively removed with base treatment, and are used to make reactive amino groups selectively available for substitution. Examples of such groups are the Fmoc (E. Atherton and R. C. Sheppard in The Peptides, S. Udenfriend, J. Meienhofer, Eds., Academic Press, Orlando, 1987, volume 9, p.1), and various substituted sulfonylethyl carbamates exemplified by the Nsc group (Samukov et al.,Tetrahedron Lett, 1994, 35:7821; Verhart and Tesser, Rec. Trav. Chim. Pays-Bas, 1987, 107:621).
Additional amino-protecting groups include but are not limited to, carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide-protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and imine- and cyclic imide-protecting groups, such as phthalimido and dithiasuccinoyl. Equivalents of these amino-protecting groups are also encompassed by the compounds and methods of the present invention.
In some especially preferred embodiments, one or more of the internucleoside linkages comprising oligomeric compounds of the invention are optionally protected phosphorothioate internucleoside linkages. Representative protecting groups for phosphorus containing internucleoside linkages such as phosphite, phosphodiester and phosphoro-thioate linkages include xcex2-cyanoethyl, diphenylsilylethyl, xcex4-cyanobutenyl, cyano p-xylyl (CPX), N-methyl-N-trifluoro-acetyl ethyl (META), acetoxy phenoxy ethyl (APE) and butene-4-yl groups. See for example U.S. Pat. No. 4,725,677 and U.S. Pat. No. Re. 34,069 (xcex2-cyanoethyl); Beaucage, S. L. and Iyer, R. P., Tetrahedron, 49 No. 10, pp. 1925-1963 (1993); Beaucage, S. L. and Iyer, R. P., Tetrahedron, 49 No. 46, pp. 10441-10488 (1993); Beaucage, S. L. and Iyer, R. P., Tetrahedron, 48 No. 12, pp. 2223-2311 (1992).
In the context of this specification, alkyl (generally C1-C20), alkenyl (generally C2-C20), and alkynyl (generally C2-C20) (with more preferred ranges from C1-C10 alkyl, C2-C10 alkenyl and C2-C10 alkynyl), groups include but are not limited to substituted and unsubstituted straight chain, branch chain, and alicyclic hydrocarbons, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl and other higher carbon alkyl groups. Further examples include 2-methylpropyl, 2-methyl-4-ethylbutyl, 2,4-diethylbutyl, 3-propylbutyl, 2,8-dibutyldecyl, 6,6-dimethyloctyl, 6-propyl-6-butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexyl and other branched chain groups, allyl, crotyl, propargyl, 2-pentenyl and other unsaturated groups containing a pi bond, cyclohexane, cyclopentane, adamantane as well as other alicyclic groups, 3-penten-2-one, 3-methyl-2-butanol, 2-cyanooctyl, 3-methoxy-4-heptanal, 3-nitrobutyl, 4-isopropoxydodecyl, 4-azido-2-nitrodecyl, 5-mercaptononyl, 4-amino-1-pentenyl as well as other substituted groups. Representative alkyl substituents are disclosed in U.S. Pat. No. 5,212,295, at column 12, lines 41-50, hereby incorporated by reference in its entirety.
Further, in the context of this invention, a straight chain compound means an open chain compound, such as an aliphatic compound, including alkyl, alkenyl, or alkynyl compounds; lower alkyl, alkenyl, or alkynyl as used herein include but are not limited to hydrocarbyl compounds from about 1 to about 6 carbon atoms. A branched compound, as used herein, comprises a straight chain compound, such as an alkyl, alkenyl, alkynyl compound, which has further straight or branched chains attached to the carbon atoms of the straight chain.
A cyclic compound, as used herein, refers to closed chain compounds, i.e. a ring of carbon atoms, such as an alicyclic or aromatic compound. The straight, branched, or cyclic compounds may be internally interrupted, as in alkoxy or heterocyclic compounds. In the context of this invention, internally interrupted means that the carbon chains may be interrupted with heteroatoms such as O, N, or S. However, if desired, the carbon chain may have no heteroatoms.
Compounds of the invention can include ring structures that include a nitrogen atom (e.g., xe2x80x94N(R1) (R2) and xe2x80x94N(R3) (R4) where (R1) (R2) and (R3) (R4) each form cyclic structures about the respective N to which they are attached). The resulting ring structure is a heterocycle or a heterocyclic ring structure that can include further heteroatoms selected from N, O and S. Such ring structures may be mono-, bi- or tricyclic, and may be substituted with substituents such as oxo, acyl, alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, amido, azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo, haloalkyl, haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro, sulfide, sulfone, sulfonamide, thiol and thioalkoxy. A preferred bicyclic ring structure that includes nitrogen is phthalimido.
In general, the term xe2x80x9cheteroxe2x80x9d denotes an atom other than carbon, preferably but not exclusively N, O, or S. Accordingly, the term xe2x80x9cheterocyclic ringxe2x80x9d denotes an alkyl ring system having one or more heteroatoms (i.e., non-carbon atoms). Heterocyclic ring structures of the present invention can be fully saturated, partially saturated, unsaturated or with a polycyclic heterocyclic ring each of the rings may be in any of the available states of saturation. Heterocyclic ring structures of the present invention also include heteroaryl which includes fused systems including systems where one or more of the fused rings contain no heteroatoms. Heterocycles, including nitrogen heterocycles, according to the present invention include, but are not limited to, imidazole, pyrrole, pyrazole, indole, 1H-indazole, xcex1-carboline, carbazole, phenothiazine, phenoxazine, tetrazole, triazole, pyrrolidine, piperidine, piperazine and morpholine groups. A more preferred group of nitrogen heterocycles includes imidazole, pyrrole, indole, and carbazole groups.
In the context of this specification, aryl groups are substituted and unsubstituted aromatic cyclic moieties including but not limited to phenyl, naphthyl, anthracyl, phenanthryl, pyrenyl, and xylyl groups. Alkaryl groups are those in which an aryl moiety links an alkyl moiety to a core structure, and aralkyl groups are those in which an alkyl moiety links an aryl moiety to a core structure.
Oligomeric compounds according to the present invention that are hybridizable to a target nucleic acid preferably comprise from about 5 to about 50 nucleosides. It is more preferred that such compounds comprise from about 8 to about 30 nucleosides, with 15 to 25 nucleosides being particularly preferred. As used herein, a target nucleic acid is any nucleic acid that can hybridize with a complementary nucleic acid-like compound. Further in the context of this invention, xe2x80x9chybridizationxe2x80x9d shall mean hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleobases. xe2x80x9cComplementaryxe2x80x9d as used herein, refers to the capacity for precise pairing between two nucleobases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. xe2x80x9cComplementaryxe2x80x9d and xe2x80x9cspecifically hybridizable,xe2x80x9d as used herein, refer to precise pairing or sequence complementarity between a first and a second nucleic acid-like oligomers containing nucleoside subunits. For example, if a nucleobase at a certain position of the first nucleic acid is capable of hydrogen bonding with a nucleobase at the same position of the second nucleic acid, then the first nucleic acid and the second nucleic acid are considered to be complementary to each other at that position. The first and second nucleic acids are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, xe2x80x9cspecifically hybridizablexe2x80x9d and xe2x80x9ccomplementaryxe2x80x9d are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule.
It is understood that an oligomeric compound of the invention need not be 100% complementary to its target RNA sequence to be specifically hybridizable. An oligomeric compound is specifically hybridizable when binding of the oligomeric compound to the target RNA molecule interferes with the normal function of the target RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed.
The oligomeric compounds of the present invention can be used in diagnostics, therapeutics and as research reagents. They can be used in pharmaceutical compositions by including a suitable pharmaceutically acceptable diluent or carrier. They further can be used for treating organisms having a disease characterized by the undesired production of a protein. The organism should be contacted with an oligonucleotide having a sequence that is capable of specifically hybridizing with a strand of nucleic acid coding for the undesirable protein. Treatments of this type can be practiced on a variety of organisms ranging from unicellular prokaryotic and eukaryotic organisms to multicellular eukaryotic organisms. Any organism that utilizes RNA-DNA transcription or RNA-protein translation as a fundamental part of its hereditary, metabolic or cellular control is susceptible to therapeutic and/or prophylactic treatment in accordance with this invention. Seemingly diverse organisms such as bacteria, yeast, protozoa, algae, all plants and all higher animal forms including warm-blooded animals, ca be treated. Further each cell of multicellular eukaryotes can be treated since they include both DNA-RNA transcription and RNA-protein translation as integral parts of their cellular activity. Many of the organelles (e.g., mitochondria and chloroplasts) of eukaryotic cells also include transcription and translation mechanisms. Thus, single cells, cellular populations or organelles can also be included within the definition of organisms that can be treated with therapeutic or diagnostic oligomeric compounds. As used herein, therapeutics is meant to include the eradication of a disease state, by killing an organism or by control of erratic or harmful cellular growth or expression.
Oligomeric compounds according to the invention can be assembled in solution or through solid-phase reactions, for example, on a suitable DNA synthesizer utilizing nucleosides, phosphoramidites and derivatized controlled pore glass (CPG) according to the invention and/or standard nucleosidic monomer precursors. In addition to nucleosides that include a novel modification of the inventions other nucleoside within an oligonucleotide may be further modified with other modifications at the 2xe2x80x2 position. Precursor nucleoside and nucleosidic monomer precursors used to form such additional modification may carry substituents either at the 2xe2x80x2 or 3xe2x80x2 positions. Such precursors may be synthesized according to the present invention by reacting appropriately protected nucleosides bearing at least one free 2xe2x80x2 or 3xe2x80x2 hydroxyl group with an appropriate alkylating agent such as, but not limited to, alkoxyalkyl halides, alkoxylalkylsulfonates, hydroxyalkyl halides, hydroxyalkyl sulfonates, aminoalkyl halides, aminoalkyl sulfonates, phthalimidoalkyl halides, phthalimidoalkyl sulfonates, alkylaminoalkyl halides, alkylaminoalkyl sulfonates, dialkylaminoalkyl halides, dialkylaminoalkylsulfonates, dialkylaminooxyalkyl halides, dialkylaminooxyalkyl sulfonates and suitably protected versions of the same. Preferred halides used for alkylating reactions include chloride, bromide, fluoride and iodide. Preferred sulfonate leaving groups used for alkylating reactions include, but are not limited to, benzenesulfonate, methylsulfonate, tosylate, p-bromobenzenesulfonate, triflate, trifluoroethylsulfonate, and (2,4-dinitroanilino)-benzenesulfonate.
Suitably protected nucleosides can be assembled into oligomeric compounds according to known techniques. See, for example, Beaucage et al., Tetrahedron, 1992, 48, 2223.
The ability of oligomeric compounds to bind to their complementary target strands is compared by determining the melting temperature (Tm) of the hybridization complex of the oligonucleotide and its complementary strand. The melting temperature (Tm), a characteristic physical property of double helices, denotes the temperature (in degrees centigrade) at which 50% helical (hybridized) versus coil (unhybridized) forms are present. Tm is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the bonds between the strands. The structure-stability relationships of a large number of nucleic acid modifications have been reviewed (Freier and Altmann, Nucl. Acids Research, 1997, 25, 4429-443).
The relative binding ability of the oligomeric compounds of the present invention was determined using protocols as described in the literature (Freier and Altmann, Nucl. Acids Research, 1997, 25, 4429-443). Typically absorbance versus temperature curves were determined using samples containing 4 uM oligonucleotide in 100 mM Na+, 10 mM phosphate, 0.1 mM EDTA, and 4 uM complementary, length matched RNA.
The in vivo stability of oligomeric compounds is an important factor to consider in the development of oligonucleotide therapeutics. Resistance of oligomeric compounds to degradation by nucleases, phosphodiesterases and other enzymes is therefore determined. Typical in vivo assessment of stability of the oligomeric compounds of the present invention is performed by administering a single dose of 5 mg/kg of oligonucleotide in phosphate buffered saline to BALB/c mice. Blood collected at specific time intervals post-administration is analyzed by HPLC or capillary gel electrophoresis (CGE) to determine the amount of the oligomeric compound remaining intact in circulation and the nature the of the degradation products.